KRT1 Antibody, FITC conjugated

Basic Understanding and Applications

What is the recommended protocol for immunofluorescence using KRT1 Antibody, FITC conjugated?

Based on validated methodology in the literature , the following protocol is recommended for optimal results:

Sample Preparation:

• For paraffin sections: Deparaffinize and rehydrate through graded alcohols

• Perform heat-mediated antigen retrieval in EDTA buffer (pH 8.0)

• For cultured cells: Fix with 4% paraformaldehyde (10 min, RT) and permeabilize with 0.1% Triton X-100 (5 min, RT)

Blocking and Antibody Incubation:

• Block with 10% goat serum in PBS for 1 hour at room temperature

• Incubate with KRT1 Antibody, FITC conjugated (5 μg/ml) overnight at 4°C

• Wash 3× with PBS (5 min each)

• Counterstain with DAPI for nuclear visualization

• Mount with anti-fade mounting medium

Visualization:

• Use fluorescence microscope with appropriate filter sets (excitation ~490 nm, emission ~515 nm)

• Include negative controls (omitting primary antibody) and positive controls (known KRT1-expressing tissue)

This protocol has been successfully used to detect KRT1 in human skin cancer tissue, highlighting its utility in pathological studies .

How should flow cytometry experiments with KRT1 Antibody, FITC conjugated be optimized?

Flow cytometry with KRT1 Antibody, FITC conjugated requires careful optimization and controls :

Cell Preparation:

• Harvest cells using appropriate methods (trypsin/EDTA for adherent cells)

• Fix with 4% paraformaldehyde (10 min, RT)

• Permeabilize with permeabilization buffer for intracellular staining

• Block with 10% normal goat serum (30 min, RT)

Antibody Staining:

• Incubate with KRT1 Antibody, FITC conjugated (1 μg per 10^6 cells) for 30 min at 20°C

• Wash 2× with PBS containing 1% BSA

Essential Controls:

• Isotype control: rabbit IgG (for rabbit polyclonal) or mouse IgG1 (for mouse monoclonal) at the same concentration

• Unlabelled sample: cells without primary antibody incubation

• Single-stain controls: when performing multiplex analysis

Analysis Considerations:

• Use appropriate gating strategies based on forward/side scatter and controls

• Analyze both percentage of positive cells and mean fluorescence intensity

• For quantitative comparisons, establish consistent instrument settings across experiments

Flow cytometry analysis of Jurkat cells has demonstrated successful detection of intracellular KRT1, with clear differentiation from control samples .

What are the key considerations for Western blot analysis using KRT1 Antibody, FITC conjugated?

Although FITC-conjugated antibodies are less commonly used for Western blotting than HRP-conjugated antibodies, they can be effectively employed with fluorescence-based detection systems. Based on protocols in the literature :

Sample Preparation:

• Lyse cells or tissues with RIPA buffer containing protease inhibitors

• Determine protein concentration (BCA or Bradford assay)

• Load appropriate amounts (10-20 μg) on SDS-PAGE gels

Blotting and Detection:

• Transfer proteins to PVDF membrane

• Block with 5% dry milk in PBST for 1 hour at room temperature

• Incubate with KRT1 Antibody, FITC conjugated (1:500 dilution) for 1 hour at room temperature

• Wash thoroughly with PBST (3× for 10 min each)

• Optional: incubate with secondary antibody (anti-fluorescein) for signal amplification

Detection Options:

• Direct fluorescence scanning with appropriate imaging systems

• For chemiluminescent detection, use anti-FITC antibody conjugated to HRP followed by ECL substrate

Expected Results:

• KRT1 appears at approximately 67 kDa

• Western blot analysis of HaCat lysates shows clear bands at both 10 μg and 20 μg protein loading

How can KRT1 Antibody, FITC conjugated be used to study KRT1's role in inflammatory conditions?

KRT1 has been implicated in inflammatory processes, particularly in ulcerative colitis and skin inflammation . Advanced methodological approaches include:

Visualization of Expression Changes:

• Compare KRT1 localization in normal versus inflamed tissues

• Quantify fluorescence intensity as a measure of expression levels

• Track subcellular redistribution during inflammatory responses

Co-localization with Inflammatory Mediators:

• Dual staining with antibodies against inflammatory cytokines (IL-1β, IL-6, TNF-α)

• Proximity ligation assays to detect protein-protein interactions

• Time-course studies to track temporal relationships

Barrier Function Assessment:

• Co-staining with tight junction proteins (occludin, ZO-1, claudin)

• Correlation of KRT1 patterns with transepithelial/transendothelial electrical resistance

• Permeability assays in conjunction with KRT1 imaging

Research has demonstrated that KRT1 treatment significantly inhibits inflammatory cytokines in DSS-induced colitis models and enhances intestinal barrier function by upregulating the expression of tight junction proteins . Furthermore, absence of Krt1 caused prenatal increases in IL-18 and S100A8/S100A9 proteins, accompanied by barrier defects, indicating its regulatory role in inflammatory networks .

How can KRT1 Antibody, FITC conjugated be utilized to investigate KRT1's function as a cell surface receptor?

Recent research has revealed that KRT1 can function as a cell-surface receptor in certain cancer cells . Sophisticated methodological approaches include:

Cell Surface Localization:

• Non-permeabilized immunofluorescence to confirm surface presence

• Surface biotinylation followed by immunoprecipitation and fluorescence detection

• Flow cytometry on non-permeabilized cells to quantify surface expression

Receptor Complex Studies:

• Co-immunoprecipitation with integrin β1, Src kinase, and RACK1

• Multi-color immunofluorescence to visualize co-localization

• FRET (Fluorescence Resonance Energy Transfer) analysis to detect molecular proximity

Functional Analysis:

• Ligand-induced internalization studies using time-lapse fluorescence microscopy

• Receptor activation assays measuring downstream signaling events

• Mutation studies to identify critical domains for receptor function

Research has shown that K1 interacts specifically with integrin β1 on neuroblastoma NMB7 cells as part of a multi-protein complex that includes tyrosine kinase Src and RACK-1, potentially serving as a platform for tyrosine kinase activation .

What methodologies can be employed to study the interaction between KRT1 and the kallikrein kinin system (KKS)?

The interaction between KRT1 and the KKS has significant implications for ulcerative colitis pathophysiology . Advanced research approaches include:

Protein Interaction Analysis:

• Proximity ligation assay to visualize KRT1 interactions with KKS components

• Co-immunoprecipitation followed by Western blotting to confirm binding

• Pull-down assays with purified components to determine direct interactions

Functional Assessment:

• Dual immunofluorescence of KRT1 with bradykinin (BK) or high molecular weight kininogen (HK)

• Quantitative analysis of expression changes following KKS activation/inhibition

• Correlation with coagulation factor XII activity measurements

In Vivo Models:

• KRT1 antibody treatment in DSS-induced colitis to assess impact on KKS activation

• Tracking KRT1 and KKS component localization during disease progression

• Assessment of intestinal barrier function markers in relation to KRT1/KKS modulation

Research demonstrates that KRT1 inhibits BK expression, suppresses inflammatory cytokines, and enhances intestinal barrier function, positioning it as a regulator of the KKS in colonic inflammation . Treatment with KRT1 protein in DSS-induced mice significantly alleviated pathological symptoms and restored intestinal epithelial barrier integrity by upregulating tight junction proteins including occludin, ZO-1, and claudin .

How can KRT1 Antibody, FITC conjugated be used in multiplex immunofluorescence studies?

Multiplex immunofluorescence allows simultaneous detection of multiple proteins, providing valuable insights into protein co-localization and interaction networks:

Fluorophore Selection and Combination:

• FITC (KRT1): excitation/emission ~490/515 nm

• Compatible fluorophores for multiplexing:

  • Cy3/TRITC: excitation/emission ~550/570 nm

  • Cy5/APC: excitation/emission ~650/670 nm

  • DAPI: excitation/emission ~358/461 nm

Protocol Optimization:

• Sequential staining approach for antibodies from the same host species

• Simultaneous staining for antibodies from different host species

• Careful titration of each antibody to minimize cross-reactivity

Controls and Validation:

• Single-stain controls to assess spectral overlap and bleed-through

• Appropriate blocking sera to minimize non-specific binding

• Isotype controls for each antibody used

Analysis Approaches:

• Confocal microscopy with sequential scanning for each fluorophore

• Quantitative co-localization analysis (Pearson's correlation, Manders' overlap coefficient)

• Spectral unmixing for closely overlapping fluorophores

This approach enables visualization of KRT1 alongside other proteins such as KRT10 (heterodimer partner), inflammatory markers, or barrier function proteins, providing spatial context for functional relationships.

How can KRT1 Antibody, FITC conjugated be used to study KRT1 in skin disorders?

KRT1 plays a crucial role in skin integrity, and mutations in KRT1 cause epidermolytic ichthyosis . Research methodologies include:

Histopathological Assessment:

• Comparison of KRT1 expression patterns between normal and affected skin

• Evaluation of KRT1 aggregation in keratinocytes with KRT1 mutations

• Co-staining with differentiation markers to assess impact on epidermal maturation

Barrier Function Studies:

• Correlation of KRT1 distribution with transepidermal water loss measurements

• Co-visualization with other barrier components (filaggrin, loricrin)

• Assessment of calcium gradient disruption in relation to KRT1 patterns

Inflammatory Response Analysis:

• Tracking IL-18 and S100A8/A9 expression in relation to KRT1 abnormalities

• Evaluation of inflammatory cell infiltration in regions with altered KRT1 expression

• Time-course studies during disease progression or treatment response

Research has shown that KRT1 mutations lead to epidermolytic ichthyosis characterized by skin erosions, hyperkeratosis, and recurrent erythema . Furthermore, absence of Krt1 in mouse models caused a prenatal increase in inflammatory mediators accompanied by barrier defects and perinatal lethality, which was partially rescued by depletion of IL-18 .

What are the applications of KRT1 Antibody, FITC conjugated in cancer research?

KRT1 has emerging roles in cancer biology, particularly as a cell-surface receptor and potential serum biomarker :

Expression Analysis:

• Comparison of KRT1 levels between normal and malignant tissues

• Correlation with tumor grade, stage, and patient outcomes

• Subcellular localization changes in cancer progression

Cell Surface Receptor Studies:

• Analysis of KRT1 interactions with integrin β1, Src, and RACK1 in cancer cells

• Assessment of receptor complex formation and signaling in different cancer types

• Functional consequences of receptor activation/inhibition on cancer cell behavior

Serum Biomarker Development:

• Flow cytometry-based detection of circulating KRT1

• Immunofluorescence visualization of KRT1 in circulating tumor cells

• Correlation with established biomarkers and disease progression

Research has demonstrated high levels of K1 in the serum of patients with hepatocellular carcinoma (HCC), with progressive increases from healthy individuals to liver cirrhosis to HCC patients . Western blot analysis revealed a ~67 kDa band in HCC and LC samples that was undetectable in samples from healthy individuals .

How can KRT1 Antibody, FITC conjugated contribute to intestinal barrier function research?

KRT1 plays a protective role in intestinal inflammation by modulating barrier integrity . Advanced methodological approaches include:

Expression Analysis in Inflammatory Bowel Disease:

• Comparison of KRT1 patterns in healthy versus diseased intestinal tissue

• Correlation with disease severity and treatment response

• Association with specific IBD phenotypes (ulcerative colitis vs. Crohn's disease)

Barrier Function Assessment:

• Co-localization with tight junction proteins (occludin, ZO-1, claudin)

• Correlation with permeability assays and transepithelial electrical resistance

• Analysis of KRT1 redistribution during barrier disruption and recovery

Therapeutic Targeting Studies:

• Monitoring effects of KRT1 protein treatment on intestinal inflammation

• Assessment of KRT1-targeting approaches on epithelial integrity

• Combination strategies with established IBD therapies

Research demonstrates that KRT1 protein treatment in DSS-induced mice significantly upregulated the expression of tight junction proteins (occludin, ZO-1, and claudin) in intestinal epithelial cells while reducing expression of the negative regulator FXIIα . These changes contributed to the repair of intestinal damage, reduction of intestinal permeability, and alleviation of inflammatory responses .

What controls should be included when using KRT1 Antibody, FITC conjugated?

Rigorous experimental design requires appropriate controls to ensure valid and interpretable results:

Antibody Controls:

• Isotype control: rabbit IgG (for rabbit polyclonal) or mouse IgG1 (for mouse monoclonal) at matching concentration

• Absorption control: pre-incubation of antibody with purified KRT1 antigen

• Secondary antibody-only control (where applicable)

Sample Controls:

• Positive control: tissues/cells known to express KRT1 (epidermis, HaCat cells)

• Negative control: tissues/cells known not to express KRT1

• KRT1 knockdown/knockout samples (where available)

Technical Controls:

• Autofluorescence control: unstained sample to assess background

• Single-color controls for spectral compensation in multiplex experiments

• Concentration gradient to determine optimal antibody dilution

Flow cytometry analysis should include unstained samples, isotype controls, and single-stain controls as demonstrated in successful KRT1 detection in Jurkat cells .

How can the specificity of KRT1 Antibody, FITC conjugated be validated?

Ensuring antibody specificity is critical for reliable research results. Comprehensive validation approaches include:

Western Blot Validation:

• Confirm single band of expected molecular weight (~67 kDa)

• Demonstrate dose-dependent signal with increasing protein concentration

• Compare results with alternative KRT1 antibodies

Genetic Approaches:

• siRNA/shRNA knockdown of KRT1 should reduce signal proportionally

• CRISPR/Cas9 knockout of KRT1 should eliminate specific signal

• Overexpression systems should show increased signal intensity

Peptide Competition:

• Pre-incubation with immunizing peptide should abolish specific staining

• Titration of competing peptide should show dose-dependent reduction in signal

• Non-relevant peptides should not affect antibody binding

Cross-Reactivity Assessment:

• Testing on multiple species to confirm cross-reactivity claims

• Evaluation on closely related keratins (KRT2, KRT3, etc.) to confirm specificity

• Application in multiple techniques to ensure consistent specificity profile

What are the critical factors affecting KRT1 Antibody, FITC conjugated performance?

Multiple factors can influence antibody performance and should be carefully controlled:

Sample Preparation:

• Fixation method and duration affect epitope accessibility

• Antigen retrieval conditions (EDTA buffer pH 8.0 recommended)

• Permeabilization parameters for intracellular targets

Antibody Factors:

• Storage conditions (4°C short-term, -20°C long-term recommended)

• Freeze-thaw cycles (should be minimized)

• Optimal working dilution (application-dependent: 1:50-200 for IF, 1:500 for WB)

Detection Parameters:

• Fluorophore photobleaching during extended imaging

• Appropriate filter sets for FITC detection (excitation ~490 nm, emission ~515 nm)

• Microscope settings (exposure time, gain, offset)

Environmental Considerations:

• Protection from light to prevent photobleaching

• Temperature consistency during incubations

• pH stability of buffers

Proper attention to these factors ensures consistent and reproducible results across experiments and between laboratories.